BMAL1 Research Articles

Treatment of acute pharyngitis in rats with season tea decoctions from traditional Chinese medicine through a synergistic and subtle regulation of ARNTL and BHLHE40

Ethnopharmacological relevanceWhile the seasonal variations in the human immune function and many infectious diseases are well-known, to develop therapeutic strategies regarding such seasonality is quite challenging. However, some traditional medical practices have already taken the seasonality into account, such as the “Season Tea” (ST) decoctions investigated in the present study. Aim of the studyWe present a study of the ST decoctions from traditional Chinese medicine, which include four formulae designed for the four seasons, aiming to investigate their pharmacological commonality and distinction. Materials and methodsA rat model of acute pharyngitis was utilized for the pharmacological study, and the effects of the ST decoctions were evaluated through histology, biomedical assays, microarray analysis, real-time quantitative PCR and Western blot. ResultsThe experimental data show that all of the four ST formulae display good pharmaceutical effects on acute pharyngitis, and circadian rhythm appears to be a significant pathway for investigating their pharmacological commonality and distinction. Specifically, while all of the four ST decoctions can regulate the circadian-rhythm-related genes ARNTL and BHLHE40, the regulation is along different directions with the modification of the supplements and the substrates in each ST formula. ConclusionThese results indicate the correlation between the acute pharyngitis and circadian rhythm, and illustrate the possibility of synergistically and subtly regulating ARNTL and BHLHE40, which is significant for relevant drug development.

The role of circadian clock gene Arntl in the winter depression-like behavior in melatonin-proficient female CBA/N mice

Seasonal affective disorder (SAD), also known as winter depression, is a subtype of depression typically manifesting in winter. Typical symptoms of SAD, such as an increased need for sleep and carbohydrate cravings associated with increased appetite and weight, are distinct from those of major depression, and the underlying mechanisms of SAD remain unclear. Although laboratory mice are generally considered non-seasonal animals, we observed depression-like behaviors in melatonin-proficient female CBA/N mice maintained under winter-mimicking conditions. Transcriptome analysis of the brains of CBA/N mice maintained under winter- and summer-mimicking conditions revealed changes in the expression of circadian clock genes, including Arntl (also known as Bmal1). We generated Arntl-deficient, melatonin-proficient CBA/N mice using the speed congenic method to examine the role of Arntl in depressive behavior. The tail suspension test in these mice revealed a depressive phenotype. These results suggested that the circadian clock gene Arntl may be involved in winter depression-like behavior.

Influence of circadian rhythm on effects induced by mechanical strain in periodontal ligament cells.

The aim of this study was to investigate the influence of mechanical strain on clock gene function in periodontal ligament (PDL) cells. Furthermore, we wanted to analyze whether effects induced by mechanical stress vary in relation to the circadian rhythm. Human PDL fibroblasts were synchronized in their circadian rhythm with dexamethasone and stretched over 24 h. Unstretched cells served as controls. Gene expression of the core clock genes were analyzed at 4 h intervals by quantitative real-time polymerase chain reaction (qRT-PCR). Time points 0 h (group SI1) and 12 h (group SI2) after synchronization served as starting points of a4 h force application period. Collagen-1α (COL-1α/Col-1α), interleukin-1β (IL1-β), and runt-related transcription factor 2 (RUNX2/Runx2) were assessed by qRT-PCR and enzyme-linked immunosorbent assay (ELISA) after 2 and 4 h. Statistical analysis comprised one-way analysis of variance (ANOVA) and posthoc tests. After synchronization, the typical pattern for clock genes was visible in control cells over the 24 h period. This pattern was significantly altered by mechanical strain. Under tensile stress, ARNTL gene expression was reduced, while Per1 and2 gene expression were upregulated. In addition, mechanical stress had adifferential effect on the expression of Col-1α and IL1‑β depending on its initiation within the circadian rhythm (group SI1 vs group SI2). For RUNX2, no significant differences in the two groups were observed. Our results suggest that mechanical stress affects the molecular peripheral oscillator of PDL cells. Vice versa, the circadian rhythm also seems to partially influence the effects that mechanical stress exerts on PDL cells.

Exploring the Therapeutic Potential of Benzoxazolone Derivatives on the Circadian Clock: An In Silico and Hypothetical Approach

Objective: Pain conditions exhibit variations linked to circadian rhythms. Circadian rhythms, regulated by clock proteins, impact melatonin levels and immune functions. Melatonin, structurally similar to indomethacin, a non-steroidal anti-inflammatory drug, serves as an alternative in pain-related conditions. Literature suggests a correlation between melatonin-dependent regulation of circadian rhythm and reductions in pain complaints. 2(3<i>H</i>)-Benzoxazolone, known for its anti-inflammatory and analgesic properties, is a promising scaffold for drug design. In this study, pharmacophore analysis focused on benzoxazolone derivatives, evaluating their impact on clock proteins, and providing insights into potential chronotherapeutic implications. Methods: Molecular docking and dynamics simulations were conducted on CLOCK:BMAL1, PER1, PER2, CRY1, and CRY2 clock proteins using benzoxazolone and its 5-substituted derivatives. Molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) calculations were employed to analyze binding free energies. Benzoxazolone derivatives, especially 5-nitro-2-benzoxazolone and 5-fluoro-2-benzoxazolone, exhibited elevated binding affinities on clock proteins compared to melatonin and indomethacin, the reference molecules. The interaction profiles and stability of complexes were maintained throughout the simulations. Results: The results suggest a regulatory impact on CRY proteins, emphasizing their role in circadian rhythm and pain modulation. In addition, the benzoxazolone ring and its derivatives, structurally resembling the core structure of melatonin and indomethacin, demonstrate promising affinities on clock proteins. Conclusion: These findings provide preliminary data and hypothetically propose benzoxazolone derivatives as potential candidates for dual functionality in analgesic activity and circadian rhythm regulation, warranting further in vitro and clinical investigations.

Phosphorylation of DNA-binding domains of CLOCK–BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

PROVIT-CLOCK: A Potential Influence of Probiotics and Vitamin B7 Add-On Treatment and Metabolites on Clock Gene Expression in Major Depression

Introduction: An increasing body of evidence suggests a strong relationship between gut health and mental state. Lately, a connection between butyrate-producing bacteria and sleep quality has been discussed. The PROVIT study, as a randomized, double-blind, 4-week, multispecies probiotic intervention study, aims at elucidating the potential interconnection between the gut’s metabolome and the molecular clock in individuals with major depressive disorder (MDD). Methods: The aim of the PROVIT-CLOCK study was to analyze changes in core clock gene expression during treatment with probiotic intervention versus placebo in fasting blood and the connection with the serum- and stool-metabolome in patients with MDD (n = 53). In addition to clinical assessments in the PROVIT study, metabolomics analyses with 1H nuclear magnetic resonance spectroscopy (stool and serum) and gene expression (RT-qPCR) analysis of the core clock genes ARNTL, PER3, CLOCK, TIMELESS, NR1D1 in peripheral blood mononuclear cells of fasting blood were performed. Results: The gene expression levels of the clock gene CLOCK were significantly altered only in individuals receiving probiotic add-on treatment. TIMELESS and ARNTL gene expression changed significantly over the 4-week intervention period in both groups. Various positive and negative correlations between metabolites in serum/stool and core clock gene expression levels were observed. Conclusion: Changing the gut microbiome by probiotic treatment potentially influences CLOCK gene expression. The preliminary results of the PROVIT-CLOCK study indicate a possible interconnection between the gut microbiome and circadian rhythm potentially orchestrated by metabolites.

Coordination between circadian neural circuit and intracellular molecular clock ensures rhythmic activation of adult neural stem cells

The circadian clock throughout the day organizes the activity of neural stem cells (NSCs) in the dentate gyrus (DG) of adult hippocampus temporally. However, it is still unclear whether and how circadian signals from the niches contribute to daily rhythmic variation of NSC activation. Here, we show that norepinephrinergic (NEergic) projections from the locus coeruleus (LC), a brain arousal system, innervate into adult DG, where daily rhythmic release of norepinephrine (NE) from the LC NEergic neurons controlled circadian variation of NSC activation through β3-adrenoceptors. Disrupted circadian rhythmicity by acute sleep deprivation leads to transient NSC overactivation and NSC pool exhaustion over time, which is effectively ameliorated by the inhibition of the LC NEergic neuronal activity or β3-adrenoceptors-mediated signaling. Finally, we demonstrate that NE/β3-adrenoceptors-mediated signaling regulates NSC activation through molecular clock BMAL1. Therefore, our study unravels that adult NSCs precisely coordinate circadian neural circuit and intrinsic molecular circadian clock to adapt their cellular behavior across the day.

Deficiency of circadian clock gene Bmal1 exacerbates noncanonical inflammasome-mediated pyroptosis and lethality via Rev-erbα-C/EBPβ-SAA1 axis

Circadian arrhythmia has been linked to increased susceptibility to multiple inflammatory diseases, such as sepsis. However, it remains unclear how disruption of the circadian clock modulates molecular aspects of innate immune responses, including inflammasome signaling. Here, we examined the potential role of the circadian clock in inflammasome-mediated responses through myeloid-specific deletion of BMAL1, a master circadian clock regulator. Intriguingly, Bmal1 deficiency significantly enhanced pyroptosis of macrophages and lethality of mice under noncanonical inflammasome-activating conditions but did not alter canonical inflammasome responses. Transcriptome analysis of enriched peritoneal myeloid cells revealed that Bmal1 deficiency led to a marked reduction in Rev-erbα expression at steady state and a significant increase in serum amyloid A1 (SAA1) expression upon poly(I:C) stimulation. Notably, we found that the circadian regulator Rev-erbα is critical for poly(I:C)- or interferon (IFN)-β-induced SAA1 production, resulting in the circadian oscillation pattern of SAA1 expression in myeloid cells. Furthermore, exogenously applied SAA1 markedly increased noncanonical inflammasome-mediated pyroptosis of macrophages and lethality of mice. Intriguingly, our results revealed that type 1 IFN receptor signaling is needed for poly(I:C)- or IFN-β-induced SAA1 production. Downstream of the type 1 IFN receptor, Rev-erbα inhibited the IFN-β-induced association of C/EBPβ with the promoter region of Saa1, leading to the reduced transcription of Saa1 in macrophages. Bmal1-deficient macrophages exhibited enhanced binding of C/EBPβ to Saa1. Consistently, the blockade of Rev-erbα by SR8278 significantly increased poly(I:C)-stimulated SAA1 transcription and noncanonical inflammasome-mediated lethality in mice. Collectively, our data demonstrate a potent suppressive effect of the circadian clock BMAL1 on the noncanonical inflammasome response via the Rev-erbα-C/EBPβ-SAA1 axis.

Functional evaluation of an electrophilic focused library to identify a covalent inhibitor against intrinsically disordered circadian clock transcription factors

In vitro screening of a focused library of compounds containing an electrophilic warhead identified N-chloroacetyl-bis(trifluoromethyl)aniline derivative 15 as a potent inhibitor of BMAL1–CLOCK heterodimer binding to an E-box DNA fragment. Kinetic analysis of thiol-reactivity demonstrated that iodoacetamide and structurally related 20 are significantly more reactive than or equally reactive as 15, respectively, whereas none inhibited BMAL1–CLOCK interaction with the E-box DNA fragment. These results suggest that 15 binds and reacts with a specific nucleophilic residue. This low-molecular-weight compound may serve as a useful lead for further development of BMAL1–CLOCK inhibitors.

Determination of the Relationship Between DNA Methylation Status of KLOTHO and ARNTL Genes With Hypertension.

Hypertension is a multifactorial chronic disease due to the interaction of environmental factors with genetic alteration. KLOTHO and ARNTL genes play an important role in the development of hypertension. Therefore, we analyzed the methylation status of KLOTHO and ARNTL genes by using methylation-sensitive high-resolution melting (MSHRM) in a total of 78 hypertensive and 49 control subjects. In this study, we could not identify a significant association between KLOTHO and ARNTL methylation and the hypertensive phenotype. Moreover, we could not find a direct association between KLOTHO and ARNTL methylation and the fasting blood sugar, triglycerides, total cholesterol, LDL-cholesterol, HDL-cholesterol, sodium (Na), creatinine (Cr), potassium (K), and urea levels in hypertensive patients. However, we found a significant difference between the methylated KLOTHO hypertensive patients and the unmethylated KLOTHO control subjects for potassium (K).

The Relationship between Clock Genes, Sirtuin 1, and Mitochondrial Activity in Head and Neck Squamous Cell Cancer: Effects of Melatonin Treatment.

The circadian clock is a regulatory system, with a periodicity of approximately 24 h, which generates rhythmic changes in many physiological processes, including mitochondrial activity. Increasing evidence links chronodisruption with aberrant functionality in clock gene expression, resulting in multiple diseases such as cancer. Melatonin, whose production and secretion oscillates according to the light-dark cycle, is the principal regulator of clock gene expression. In addition, the oncostatic effects of melatonin correlate with an increase in mitochondrial activity. However, the direct links between circadian clock gene expression, mitochondrial activity, and the antiproliferative effects of melatonin in cancers, including head and neck squamous cell carcinoma (HNSCC), remain largely unknown. In this study, we analyzed the effects of melatonin on HNSCC cell lines (Cal-27 and SCC9), which were treated with 500 and 1000 µM melatonin. We found that the antiproliferative effect of melatonin is not mediated by the Bmal1 clock gene. Additionally, high doses of melatonin were observed to result in resynchronization of oscillatory circadian rhythm genes (Per2 and Sirt1). Surprisingly, the resynchronizing effect of melatonin on Per2 and Sirt1 did not produce alterations in the oscillation of mitochondrial respiratory activity. These results increase our understanding of the possible antiproliferative mechanisms in melatonin in the treatment of head and neck squamous cell carcinoma and suggest that its antiproliferative effects are independent of clock genes but are directly related to mitochondrial activity.

IMS-Bmal1-/- mice show evident signs of sarcopenia that are counteracted by exercise and melatonin therapies.

Sarcopenia is an age-related disease characterized by a reduction in muscle mass, strength, and function and, therefore, a deterioration in skeletal muscle health and frailty. Although the cause of sarcopenia is still unknown and, thus, there is no treatment, increasing evidence suggests that chronodisruption, particularly alterations in Bmal1 clock gene, can lead to those deficits culminating in sarcopenia. To gain insight into the cause and mechanism of sarcopenia and the protective effect of a therapeutic intervention with exercise and/or melatonin, the gastrocnemius muscles of male and female skeletal muscle-specific and inducible Bmal1 knockout mice (iMS-Bmal1-/- ) were examined by phenotypic tests and light and electron microscopy. Our results revealed a disruption of the normal activity/rest rhythm, a drop in skeletal muscle function and mass, and increased frailty in male and female iMS-Bmal1-/- animals compared to controls. A reduction in muscle fiber size and increased collagenous tissue were also detected, accompanied by reduced mitochondrial oxidative capacity and a compensatory shift towards a more oxidative fiber type. Electron microscopy further supports mitochondrial impairment in mutant mice. Melatonin and exercise ameliorated the damage caused by loss of Bmal1 in mutant mice, except for mitochondrial damage, which was worsened by the latter. Thus, iMS-Bmal1-/- mice let us to identify Bmal1 deficiency as the responsible for the appearance of sarcopenia in the gastrocnemius muscle. Moreover, the results support the exercise and melatonin as therapeutic tools to counteract sarcopenia, by a mechanism that does not require the presence of Bmal1.

Downregulation of ARNTL in renal tubules of diabetic db/db mice reduces kidney injury by inhibiting ferroptosis

BackgroundThe prevalence of ferroptosis in diabetic kidney tubules has been documented, yet the underlying mechanism remains elusive. The aim of this study was to ascertain the pivotal gene linked to ferroptosis and establish a novel target for the prevention and management of diabetic kidney disease (DKD). MethodsTranscriptomics data (GSE184836) from DKD mice (C57BLKS/J) were retrieved from the GEO database and intersected with ferroptosis-related genes from FerrDb. Then, differentially expressed genes associated with ferroptosis in the glomeruli and tubules were screened. Gene ontology analysis and protein-protein interaction network construction were used to identify key genes. Western blotting and real-time quantitative polymerase chain reaction were employed to validate the expression in the same model. Aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL) expression in patients and mice with DKD was assessed using immunohistochemistry staining. ARNTL knockdown in C57BLKS/J mice was established and plasma malonaldehyde, superoxide dismutase, and renal pathology were analyzed. The efficacy of ARNTL knockdown was evaluated using proteomics analysis. Mitochondrial morphology was observed using transmission electron microscopy. ResultsARNTL was screened by bioinformatics analysis and its overexpression verified in patients and mice with DKD. ARNTL knockdown reduced oxidative stress in plasma. Kidney proteomics revealed that ferroptosis was inhibited. The reduction of the classic alteration in mitochondrial morphology associated with ferroptosis was also observed. Gene set enrichment analysis demonstrated that the downregulation of the TGFβ pathway coincided with a decrease in collagen protein and TGFβ1 levels. ConclusionsThe ferroptosis-associated gene ARNTL is a potential target for treating DKD.

Circadian gene ARNTL initiates circGUCY1A2 transcription to suppress non-small cell lung cancer progression via miR-200c-3p/PTEN signaling

BackgroundAs a subclass of endogenous stable noncoding RNAs, circular RNAs are beginning to be appreciated for their potential as tumor therapeutics. However, the functions and mechanisms by which circRNAs exert protective functions in non-small cell lung cancer (NSCLC) remain largely elusive.MethodsThe prognostic role of circGUCY1A2 was explored in lung adenocarcinoma specimens. The overexpressed and knockdown plasmids were used to evaluate the effect of circGUCY1A2 on NSCLC cell proliferation and apoptosis efficacy. Luciferase reporter system is used to prove that circGUCY1A2 could bind to miRNA. Chip-PCR was used to prove that circGUCY1A2 could be initiated by transcription factors ARNTL. Subcutaneous tumorigenicity grafts models were established to validate findings in vivo.ResultsThe expression of circGUCY1A2 were significantly reduced (P < 0.001) and negatively correlated with tumor size (P < 0.05) in non-small cell lung cancer (NSCLC). CircGUCY1A2 upregulation promoted apoptosis and inhibits cell proliferation and growth of subcutaneous tumorigenicity grafts in nude mice (P < 0.01). In addition, intra-tumor injection of pLCDH-circGUCY1A2 inhibited tumor growth in patient-derived NSCLC xenograft models (PDX). Mechanism studies showed that circGUCY1A2 could act as a sponge to competitively bind miR-200c-3p, promote PTEN expression, and thereby inhibit PI3K/AKT pathway. In addition, we found that the circadian gene ARNTL, which was reduced in NSCLC and prolonged the overall survival of patients, could bind to the promoter of circGUCY1A2, thereby increasing its expression.ConclusionsThis study is an original demonstration that ARNTL can inhibit the development of lung adenocarcinoma through the circGUCY1A2/miR-200c-3p/PTEN axis, and this finding provides potential targets and therapeutic approaches for the treatment of lung adenocarcinoma.

Research progresses on the effects of heavy metals on the circadian clock system.

Environmental pollution with heavy metals is widespread, thus increasing attention has been paid to their toxic effects. Recent studies have suggested that heavy metals may influence the expression of circadian clock genes. Almost all organs and tissues exhibit circadian rhythms. The normal circadian rhythm of an organism is maintained by the central and peripheral circadian clock. Thus, circadian rhythm disorders perturb normal physiological processes. Here, we review the effects of heavy metals, including manganese, copper, cadmium, and lead, on four core circadian clock genes, i.e.,ARNTL, CLOCK, PER, and CRY genes.

Association of the circadian rhythm gene &lt;i&gt;ARNTL/BMAL1&lt;/i&gt; with personal anxiety among people aged 25–64 (WHO international program “MONICA-psychosocial (MOPSY)”)

Objective: to study associations between personal anxiety (PA) and single nucleotide polymorphism rs2278749 of the ARNTL gene among individuals aged 25–64 years living in Novosibirsk.Material and methods. Under the WHO program “MONICA-psychosocial (MOPSY)”, a random representative sample of the population aged 25–64 years in Novosibirsk was studied (725 men, mean age – 43.4±0.4 years, response – 71.3%; 710 women, mean age – 44.8±0.4 years, response – 72%). The general examination was carried out according to standard methods included in the protocol of the WHO program. To assess PA, a form of Spielberger self-assessment scales was proposed. Every second respondent underwent genotyping of the studied polymorphisms of the ARNTL gene.Results. The C/C genotype of the ARNTL gene was found in the general population in 60.7% of individuals (in 61.2% of men and 60.5% of women); the C/T genotype was found in 34.1% of individuals (in 35.1% of men and 33.5% of women) and the T/T genotype in the general population was found in 5.2% of individuals (in 3.7% of men and 6% women). The probability of PA development among carriers of the CT+TT genotypes of the ARNTL gene was 2 times higher (p&lt;0.05) in the general population and 2.4 times (p&lt;0.05) among women; among T allele carriers, it was 1.8 times higher (p&lt;0.05) in the population and 2.1 times (p&lt;0.05) among women. Carriers of the C/T genotype of the ARNTL gene were 30.3% more likely to believe that they almost always take everything too personally (p=0.024). Carriers of the C/T genotype (21%) almost always, and carriers of the T/T genotype (27.8%) often would like to be as happy as others (p=0.031). The answer “I often feel satisfaction” prevailed (38.2%) among the carriers of the C/C genotype, and the answer “Almost never feel satisfaction” among the carriers of the C/T genotype (5.9%) of the ARNTL gene (p=0.044) .Conclusion. It was found that the C/C genotype of the ARNTL gene was the most common in the population; the probability of PA occurrence among carriers of CT+TT genotypes, carriers of the T allele is 2 times higher than in carriers of other genotypes of the ARNTL gene.

Defining diurnal fluctuations in mouse choroid plexus and CSF at high molecular, spatial, and temporal resolution

Transmission and secretion of signals via the choroid plexus (ChP) brain barrier can modulate brain states via regulation of cerebrospinal fluid (CSF) composition. Here, we developed a platform to analyze diurnal variations in male mouse ChP and CSF. Ribosome profiling of ChP epithelial cells revealed diurnal translatome differences in metabolic machinery, secreted proteins, and barrier components. Using ChP and CSF metabolomics and blood-CSF barrier analyses, we observed diurnal changes in metabolites and cellular junctions. We then focused on transthyretin (TTR), a diurnally regulated thyroid hormone chaperone secreted by the ChP. Diurnal variation in ChP TTR depended on Bmal1 clock gene expression. We achieved real-time tracking of CSF-TTR in awake TtrmNeonGreen mice via multi-day intracerebroventricular fiber photometry. Diurnal changes in ChP and CSF TTR levels correlated with CSF thyroid hormone levels. These datasets highlight an integrated platform for investigating diurnal control of brain states by the ChP and CSF.

Abstract 3715: Pharmacological targeting of circadian clock genes reveals regulatory mechanisms of E-box regulated genes in cancer

Abstract Transcription factors (TFs) are key regulators of homeostasis and cancer. Recent advances in pharmacological targeting of TFs makes them valuable targets in various diseases, but systems-level understanding of the function and regulation of TFs from different families in cancers remains lacking. Our lab and collaborators have been developing new small molecules to modulate the functions of key regulators of the circadian clock BMAL1 and CLOCK, which are bHLH family TFs that activate gene expression by binding to a DNA motif called E-box, with a consensus sequence of CANNTG. E-boxes are ubiquitous in the genome and broadly regulate essential physiological processes of the cell, but their molecular biology in cancer is largely unknown. In the current work we tested the ability of small molecules to regulate the expression of BMAL1 and CLOCK target genes. Across different cell lines of various cancer types, we found that stabilization of one of the negative regulators of CLOCK, CRY2, by our novel compound called SHP1705, is the most consistent to suppress canonical BMAL1 target genes. GO and GSEA analysis of RNA-seq data shows that SHP1705 also significantly changed most core regulators of circadian rhythms as well as other E-box-binding proteins. However, SHP1705 has IC50 values above 10µM in most tested cells, creating a challenge for efficacy in a clinical setting. Because BMAL1 requires the proteasome to sustain the turnover of a transcription burst, we hypothesized proteasome inhibitors (PI) MG132 and carfilzomib may synergize with SHP1705. Consequently, we found strong synergy with both PIs. Analysis of TF-target genes in combination-treated cells reveals significant changes in expression profiles, suggesting that these genes are regulated by E-box, and other related/alternative promoter elements: CCAAT-box, CG-rich motifs, and ATGGC. Co-enrichment of these elements revealed the components of E-box containing promoters and showed potential properties of E-box-regulated gene expression. Co-existence of these motifs in the same proximal promoter also potentially allows RNAPII multiple choices of alternative promoters and could promote expression of variants in cancer cells. Our results support the necessity of inhibiting the whole promoter and provide an example of how to approach designer pharmacological strategies to achieve this purpose. Citation Format: Yuanzhong Pan, Heinz-Josef Lenz, Evanthia Roussos Torres, Steve A. Kay. Pharmacological targeting of circadian clock genes reveals regulatory mechanisms of E-box regulated genes in cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3715.

BMAL1 and CLOCK control polarization and lumen formation during mammary alveologenesis in 3D culture

Circadian clock disruption decreased mammary development and impaired lactation in late gestation cows and mice. Transcriptional targets of the circadian clock genes BMAL1 and CLOCK include the cell-cell and cell-ECM proteins e-cadherin (CDH1) and zonula occludens-1 (ZO-1). The polarization and lumen formation of milk-producing acini is dependent on these junctional proteins. We hypothesized that if BMAL1 and CLOCK were disrupted, mammary epithelial cells (HC11) will have a reduced ability to form acini. Our objective was to measure the effect of BMAL1 gene deletion (BMAL1-KO) and CLOCK protein reduction (shCLOCK) in HC11 cells on the formation of acini and expression of CDH1 and ZO-1 in three-dimensional (3D) cultures. Cells were plated on Matrigel, embedded, and cultured to create 3D structures. ImageJ software was used to quantify the acini and found that the BMAL1-KO and shCLOCK lines formed fewer and smaller acini (p &lt; 0.05). Confocal microscopy revealed that CDH1 was located along lateral membranes and ZO-1 was apically located in all three lines. Levels of CDH1 and ZO-1 were greater (p &lt; 0.05) per unit volume of cell in the shCLOCK cell line. Live/dead staining of cells found little to no cell death across three lines. Transmission electron microscopy (TEM) of acini indicated less polarization of cells within BMAL1-KO and shCLOCK acini. Polarization of cells is required for formation of 3D structures and requires coordination of temporal-spatial cues. Disruption of circadian clocks results disturbs temporal organization of cellular processes and causes a reduced ability of cells to polarize and form acini.

Level of constitutively expressed BMAL1 affects the robustness of circadian oscillations

The circadian rhythm is a biological oscillation of physiological activities with a period of approximately 24 h, that is driven by a cell-autonomous oscillator called the circadian clock. The current model of the mammalian circadian clock is based on a transcriptional-translational negative feedback loop in which the protein products of clock genes accumulate in a circadian manner and repress their own transcription. However, several studies have revealed that constitutively expressed clock genes can maintain circadian oscillations. To understand the underlying mechanism, we expressed Bmal1 in Bmal1-disrupted cells using a doxycycline-inducible promoter and monitored Bmal1 and Per2 promoter activity using luciferase reporters. Although the levels of BMAL1 and other clock proteins, REV-ERBα and CLOCK, showed no obvious rhythmicity, robust circadian oscillation in Bmal1 and Per2 promoter activities with the correct phase relationship was observed, which proceeded in a doxycycline-concentration-dependent manner. We applied transient response analysis to the Bmal1 promoter activity in the presence of various doxycycline concentrations. Based on the obtained transfer functions, we suggest that, at least in our experimental system, BMAL1 is not directly involved in the oscillatory process, but modulates the oscillation robustness by regulating basal clock gene promoter activity.